High yield algal biomass production without concentrated CO2 supply under open pond conditions

ABSTRACT

Methods and systems for efficient culturing of algae in open ponds are described.

RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.15/498,621, filed under 35 U.S.C. § 111(a) on Apr. 27, 2017, nowallowed; which claims priority to U.S. Provisional Application No.62/328,296, filed under 35 U.S.C. § 111(b) on Apr. 27, 2016. The entiredisclosures of all the aforementioned applications are herebyincorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumberCHE-1230609 awarded by the National Science Foundation, and Grant NumberDE-EE0005993 awarded by the United States Department of Energy. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Renewable energy from biomass (bioenergy) can mitigate anthropogenic CO₂emissions due to reduced use of fossil energy. Cultivation of microalgaefor bioenergy could be a superior and sustainable alternative toterrestrial energy crops, due to the fast growth rates of microalgae aswell as their ability to grow on waste waters and marginal lands. Whilethe potential of microalgae has been well-appreciated, present methodsof cultivation pose significant hurdles in the way of economicalproduction. Two methods of cultivation are closed photo-bio reactors andopen-pond systems. Of these, open-pond systems are robust forlarge-scale algal cultivation.

Microalgae cultivation in open ponds is usually attempted in anautotrophic mode (i.e., photosynthetic carbon fixation) using mesophiles(viz., algae that grow in a near neutral pH environment). To achievehigh photosynthesis rates, availability of dissolved inorganic carbon(DIC) (i.e., dissolved CO₂ and HCO₃ ⁻) is generally crucial apart fromlight. Unfortunately, under mesophilic conditions, slow kinetics ofatmospheric CO₂ absorption lead to limited DIC availability for biomassgrowth. Consequently, to increase the DIC, different approaches havebeen attempted. One of these approaches involves sparging raw flue gasor more concentrated CO₂ into the ponds. Providing concentrated CO₂(either as flue gas or more concentrated CO₂) further for algae cultureproves to be expensive, due to the high costs of CO₂ capture at theemission source using absorbents, regeneration of the absorbents, CO₂transportation to algal ponds, the costs associated with its temporarystorage, and incomplete uptake by the open pond culture medium.

Some alternatives to this approach involve contacting the sorbentsolution containing the absorbed CO₂ with the open pond culture mediumdirectly to strip the DIC into the culture, thus achieving costreductions through elimination of sorbent regeneration and CO₂ storagesteps. However, a drawback to these approaches is that they areconstrained by (i) proximate availability of flue gas or other highconcentration CO₂ sources, and (ii) the energy and infrastructure burdento deliver CO₂ over long distances, as well as its distribution into thepond-medium. It has been estimated that microalgae cultivation systemsthat are constrained by the availability of flue gases (in addition tolow-slope barren lands and favorable climates) could achieve less than10% of the Department of Energy's 2030 advanced fuel targets. Inaddition, it is believed that nearly 65% of cultivation-related variableoperating costs are associated with recovery of CO₂ from flue gas anddelivery to ponds (of a total operating cost of $144 per ton of dryalgae, approximately $91 are attributable to CO₂ delivery to ponds). Interms of overall costs of cultivation (excluding harvesting costs, butincluding costs to service capital for pond construction), CO₂ supplycontributes nearly $100 to the minimum biomass selling price (MBSP) of$400/ton of dry algae.

When “high-value” algae-based end-products are targeted (instead offuel), an alternate strategy that could be justified is mixotrophiccultivation (i.e., supplementing CO₂-derived inorganic carbon withorganic carbon such as glucose) to improve the biomass yield. However,in open pond cultivation systems, mixotrophic mode cultivation raisesadditional issues. For example, at the pH conditions conducive formesophilic algal growth, simultaneous growth of predatorymicro-organisms is also supported by the organic carbon source, leadingto algae “culture-crash”. Thus, there is a need for new and improvedmethods and systems for the culturing of algae.

SUMMARY OF THE INVENTION

Provided is a method for cultivation of algae without requiringconcentrated CO₂ inputs. The cultures are grown at high pH (>9.5), whichallows rapid absorption of atmospheric CO₂ and permits high growth rates(>10 g/m²/d).

In one aspect, provided is a method for culturing algae, the methodcomprising culturing alkaliphilic algae in an open pond medium having apH above 9.5, and incorporating into the open pond medium an inorganiccarbon buffer sufficient to allow increased fixation of atmospheric CO₂into the open pond medium, where the open pond medium is free from anyconcentrated supply of CO₂, and no concentrated source of CO₂ is used tosupply carbon to the open pond medium. In certain embodiments, theinorganic carbon buffer comprises either a NaHCO₃/Na₂CO₃ mixture or aKHCO₃/K₂CO₃ mixture. In particular embodiments, the NaHCO₃/Na₂CO₃mixture or KHCO₃/K₂CO₃ mixture is incorporated at a concentrationranging from about 7 mM to about 1 M. In certain embodiments, the pH isat least about 9.9. In certain embodiments, the method further comprisesincorporating glucose or other sugars or carboxylic acids into the openpond medium. In certain embodiments, the algae achieve growth rateshigher than 10 g/m²/d. In certain embodiments, the algae comprise aChlorella sp., Dunaliella sp., Synechocystic sp., Cyanothece sp.,Microcoleus sp., Euhalothece sp., or Spirulina sp. strain.

In certain embodiments, the method further comprises incorporating Caand/or Mg into the open pond at a concentration of less than 7 mg/L. Incertain embodiments, the low Ca and Mg lead to production of biomasswith higher carbohydrate and lipid content. In particular embodiments,the Ca is incorporated into the open pond at a concentration of lessthan 1.5 mg Ca/L. In particular embodiments, the Mg is incorporated intothe open pond at a concentration of less than 0.5 mg Mg/L.

In certain embodiments, the method further comprises circulating thealgae within the open pond medium. In certain embodiments, the methodfurther comprises harvesting biomass from the cultured algae andrecovering remnant media. In particular embodiments, the remnant mediais recycled in a second open pond medium. In particular embodiments, themethod further comprises converting the harvested biomass to one or morefuels. In particular embodiments, the converting comprises hydrothermalliquefaction to produce biocrude. In particular embodiments, thebiocrude has a N content of less than 4%.

In certain embodiments, the method further comprises regulating nitrogeninput in the open pond medium, in a range from about 5 mg/L to about 27mg/L, so as to modulate the biochemical composition of the microalgae.

In certain embodiments, the open pond medium has a salinity in the rangeof from about 10 g/L to about 30 g/L, a pH greater than 10.0, and analkalinity of up to about 1 M.

In certain embodiments, the method further comprises improvingphycocyanin production by increasing one or more of biomassconcentration, nitrogen concentration, and salinity in the open pondmedium.

In another aspect, provided herein is an open pond system comprising amedium having a pH above 9.5 and exposed to solar radiation, aninorganic carbon buffer in the medium, and alkaliphilic algae in themedium, where the open pond system is free from any unnatural orconcentrated CO₂ supply. In certain embodiments, the pH is at leastabout 9.9.

In certain embodiments, the open pond system further comprises anorganic substrate in the medium. In particular embodiments, the organicmedium comprises glucose or other sugars or carboxylic acids. In certainembodiments, the inorganic carbon buffer comprises either aNaHCO₃/Na₂CO₃ mixture or a KHCO₃/K₂CO₃ mixture. In particularembodiments, the NaHCO₃/Na₂CO₃ mixture or KHCO₃/K₂CO₃ mixture isincorporated at a concentration ranging from about 7 mM to about 1 M.

In certain embodiments, the algae comprises a Chlorella sp., Dunaliellasp., Synechocystic sp., Cyanothece sp., Microcoleus sp., Euhalothecesp., or Spirulina sp. strain. In certain embodiments, the open pondsystem further comprises a water-moving device configured to circulatethe medium within the open pond system.

In certain embodiments, the medium further comprises Ca and/or Mg at aconcentration of less than 7 mg/L.

In certain embodiments, the medium further comprises one or morenutrients selected from the group consisting of: NaNO₃, MgSO₄, CaCl₂,NaCl, ferric ammonium citrate, H₃BO₃, MnCl₂, ZnCl₂, CuCl₂, Na₂MoO₄,CoCl₂, NiCl₂, V₂O₅, and KBr.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIGS. 1A-1B: Graphs showing pH-dependent changes in bicarbonateconcentrations and mass transfer driving force (FIG. 1A), and inenhancement factor and CO₂ flux (FIG. 1B).

FIG. 2: Illustration depicting the cellular DIC transport and fixationmechanisms in alkaline media.

FIGS. 3A-3C: Graphs showing cell growth, biomass productivity, rapidlight curve, and nitrate utilization parameters of SLA-04 grown underdifferent inorganic carbon conditions (Experiment A). FIG. 3A: Cell dryweight. FIG. 3B: Biomass productivity. FIG. 3C: Rapid light curveshowing measurement of changes in electron transfer rate (ETR) withincreasing incident photon intensity.

FIGS. 4A-4B: Graphs showing pH change during SLA-04 growth (FIG. 4A),and for Experiment A (FIG. 4B).

FIGS. 5A-5C: Graphs showing cell growth, biomass productivity, rapidlight curve, and nitrate utilization parameters of SLA-04 grown underdifferent pH and inorganic carbon conditions. FIG. 5A: Cell dry weight.FIG. 5B: Biomass productivity. FIG. 5C: Rapid light curve.

FIGS. 6A-6B: Graphs showing pH change during SLA-04 growth (FIG. 6A) andcarbon balance of SLA-04 growth system (FIG. 6B).

FIG. 7: Graph showing CO₂ absorption under abiotic (without algae)conditions.

FIGS. 8A-8B: Results of phototrophic SLA-04 outdoor (750 L) raceway pondcultivation (FIG. 8A), and carbon balance for cultures grown underphototrophic conditions (FIG. 8B).

FIG. 9: Results of mixotrophic cultivation of SLA-04 outdoor (750 L)raceway pond cultivation.

FIG. 10: Graph showing biomass productivity under fresh and recyclingmedia conditions.

FIG. 11: Graph showing the effect of initial nitrate concentration onnitrogen percent of harvested biomass.

FIG. 12: SLA-04 productivities in high and low Ca/Mg media.

FIGS. 13A-13B: Biomass productivity for cultures grown in different Nconcentrations (FIG. 13A), and chlorophyll concentration for culturesgrown in different N concentrations (FIG. 13B), given biomassproductivity for two days' period.

FIGS. 14A-14F: Effect of media N input on SLA-04 cultures in 30 Lreactors. FIG. 14A shows biomass productivity. FIG. 14B shows biomassproduced/chlorophyll (g/g). FIG. 14C shows chlorophyll concentration.FIG. 14D shows nitrogen content in biomass (%). FIG. 14E shows maximumquantum yield (F_(V)/F_(M)). FIG. 14F shows FAME content.

FIGS. 15A-15B: Effect of media N input on SLA-04 cultures cultivated in1100 L reactors. FIG. 15A shows the results from batch 1, and FIG. 15Bshows the results from batch 2.

FIGS. 16A-16B: Biomass productivity (FIG. 16A), and maximum quantumyield of cultures grown with and without salinity conditions (FIG. 16B).

FIGS. 17A-17D: Cell dry weight (FIG. 17A), biomass productivity (FIG.17B), chlorophyll content (FIG. 17C), and chlorophyll a/b ratio ofcultures grown with salinity (0-30 g/L) (FIG. 17D).

FIGS. 18A-18E: Cell dry weight (FIG. 18A), biomass productivity (FIG.18B), phycocyanin content (FIG. 18C), chlorophyll content (FIG. 18D),and chlorophyll a/b ratio of cultures grown with salinity (0-30 g/L)(FIG. 18E).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents, and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents, and published patentspecifications are hereby incorporated by reference into the presentdisclosure in their entirety to more fully describe the state of the artto which this invention pertains.

Provided is a method that can cultivate microalgae under high pH andalkalinity conditions at high productivity without a supply ofconcentrated CO₂ in any form. Consequently, the method reducesproduction costs up to 25%. Furthermore, the method allows for openponds to be used in geographic areas not co-located with a source ofCO₂. In other words, the method herein alleviates the need for an openpond to be in proximity to a flue gas source. In order to be able toachieve high growth rates using atmospheric CO₂ alone, media design iskey. In general, the media should have a high pH to drive atmosphericCO₂ into solution at high rates, and should have a sufficient inorganiccarbon “buffer” to allow rapid rates of CO₂ fixation. The algae strainmust also be capable of withstanding both the high pH and high inorganiccarbon concentrations in the media. The high pH conditions allow the useof organic carbon (e.g., glucose or other sugars or carboxylic acids) tobe used as a substrate in low-cost open ponds, without concern of aculture crash because most bacteria do not survive in the high pHconditions of the medium. Further, the method produces lower amounts ofnitrogen in the algae, which is advantageous for biofuel production fromthe harvested biomass.

In accordance with the present disclosure, the cultivation ofalkaliphilic algae under appropriately tailored media conditions caneliminate most of the obstacles encountered with mesophilic algaecultivation in open-ponds. These include (1) the need to situate openponds close to a CO₂ emission source, (2) costs associated with CO₂concentration, and (3) the energy and infrastructure costs associatedwith the supply of CO₂ for commodity-scale biomass production. It isdemonstrated herein that the high pH media conditions of alkaliphilicalgae make it possible to carry out open-pond cultivation in“mixotrophic mode” without culture crash and without detrimentalmicrobial contamination. These advantages are derived from the abilityof highly alkaline solutions to efficiently absorb atmospheric CO₂, andthe inability of predatory microorganisms to survive under alkalineconditions. Moreover, with this method, after harvesting the microalgae,the aqueous medium which has high inorganic carbon and other nutrientscan be recycled indefinitely without compromising the algal growth. Inaddition, the cultivation conditions reduce the nitrogen content of thebiomass—an aspect that is highly advantageous for producing low nitrogencontent biofuels from biomass intermediates (such as throughhydrothermal liquefaction). Furthermore, cultivation of alkaliphilicmicroalgae under high salinity environment promotes the production ofphycocyanin, a high value pigment.

Alkaliphiles are organisms that thrive at high pH values (>9.5). Assuch, the cultivation medium is at an initial pH ˜10 or higher, andcontains high concentrations of inorganic carbon, up to 60-100 mM in theform of added NaHCO₃/Na₂CO₃ and/or KHCO₃/K₂CO₃ Alkaline solutions areespecially effective in absorbing “atmospheric CO₂” and sustaining theproductivity of algae, without the need for a concentrated CO₂ sourceand the infrastructure for CO₂ distribution. Simultaneously, the liquidphase equilibrium between OH⁻, CO₃ ²⁻, and HCO₃ ⁻ allows the solution tocontain high concentrations of HCO₃ ⁻, which is a DIC form usable bymicroalgae through carbonic anhydrases.

The mass transfer flux of CO₂ (J_(CO) ₂ ) from the atmosphere intohighly alkaline bulk media is determined by (i) the driving forceestablished by the concentration gradient of CO₂ between the gas-liquidinterface ([CO*_(2(aq))]) and the bulk liquid ([CO_(2(aq)) ^(bulk)]),(ii) an “enhancement factor”(E) that accounts for the enhancement ofmass transfer rates due to reaction between (the acidic) CO₂ and thehigh concentration of hydroxyl ions in the mass transfer boundary layer,and (iii) the mass transfer coefficient (k_(L)):J _(CO) ₂ =k _(L) ·E·([CO*_(2(aq))]−[CO_(2(aq)) ^(bulk)])  (1)where k_(L) is is the physical mass transfer coefficient (m/h).

At the interface with air, the liquid-phase concentration of CO₂([CO*_(2(aq))]) is determined by the concentration of CO₂ in air(assumed to be 387 ppm) and the Henry's constant for CO₂([CO*_(2(aq))]=0.013 mM). In the bulk, the aqueous CO₂ concentration([CO_(2(aq)) ^(bulk)]) is determined by the simultaneous equilibriaestablished among reactions shown in Equations 2, 3, and 4 coupled tothe electro-neutrality (total alkalinity) requirement shown in Equation5:

$\begin{matrix}{{{{CO}_{2\mspace{11mu}{({aq})}}^{bulk} + {OH}^{-}}\overset{k_{11}}{\underset{k_{12}}{\rightleftarrows}}{HCO}_{3}^{-}},{{{with}\mspace{14mu} K_{1}} = {\frac{k_{11}}{k_{12}} = {\frac{\left\lbrack {HCO}_{3}^{-} \right\rbrack}{\left\lbrack {CO}_{2\mspace{11mu}{({aq})}}^{bulk} \right\rbrack\left\lbrack {OH}^{-} \right\rbrack} = {4.5 \times 10^{7}\frac{L}{mol}\mspace{14mu}{at}\mspace{14mu} 25{^\circ}\mspace{11mu}{C.}}}}}} & (2) \\{{{{HCO}_{3}^{-} + {OH}^{-}}\overset{k_{21}}{\underset{k_{22}}{\rightleftarrows}}{{CO}_{3}^{2 -} + {H_{2}O}}},{with},\mspace{14mu}{K_{2} = {\frac{k_{21}}{k_{22}} = {\frac{\left\lbrack {CO}_{3}^{2 -} \right\rbrack}{\left\lbrack {HCO}_{3}^{-} \right\rbrack\left\lbrack {OH}^{-} \right\rbrack} = {4.9 \times 10^{3}\frac{L}{mol}\mspace{14mu}{at}\mspace{14mu} 25{^\circ}\mspace{11mu}{C.}}}}}} & (3) \\{{{H^{+} + {OH}^{-}}\overset{k_{31}}{\underset{k_{32}}{\rightleftarrows}}{H_{2}O}},{with},\mspace{14mu}{K_{w} = {\frac{k_{32}}{k_{31}} = {{\left\lbrack H^{+} \right\rbrack\left\lbrack {OH}^{-} \right\rbrack} = {0.92 \times 10^{- 14}\frac{{mol}^{2}}{L^{2}}\mspace{14mu}{at}\mspace{14mu} 25{^\circ}\mspace{11mu}{C.}}}}}} & (4) \\{\mspace{79mu}{{TA} = {\left\lbrack {HCO}_{3}^{-} \right\rbrack + {2\left\lbrack {CO}_{3}^{2 -} \right\rbrack} + \left\lbrack {OH}^{-} \right\rbrack}}} & (5)\end{matrix}$where TA is the “total alkalinity” of the system, and can be measuredindependently via titration. The equilibrium constant (K) values arefrom the literature. The plots in FIG. 1A (estimated by solvingEquations 2-5) show that in a high alkalinity medium and at pH 10.2,approximately 35 mM HCO₃ ⁻ is present along with a high CO₂ masstransfer driving force.

The pH driven enhancement factor (E) can significantly increase masstransfer rates in high pH media. For high alkalinity solutions reactingwith small concentrations of CO₂, the concentrations of CO₃ ²⁻ and HCO₃⁻ can be considered essentially invariant in the mass transfer boundarylayer. At these conditions, the enhancement factor can be estimated fromthe solution of the ordinary differential equations that describe theone-dimensional mass transport of CO₂ via the reaction shown in Eq. 2.The expression for E can be given as:

E = 1 + OH - · HCO 3 - · K 1 · [ OH - ] CO 2 ⁢ ⁢ ( K 1 · CO 2 ⁢ ( aq ) * ·HCO 3 - + OH - ) ( 6 )where, the subscripted

's represent diffusion coefficients of the various dissolved species. Asseen from Eq (6), at a constant temperature, E is function of solutionpH only (see computed values in FIG. 1B; E at pH 10.2=40).

The physical mass transfer coefficient of CO₂ (k_(L)) in open racewayponds has been previously estimated to be 0.1 m/h. At this k_(L), theCO₂ mass transfer flux values can be estimated as a function of pH usingcomputed values of E and ([CO*_(2(aq))]−[CO_(2(aq)) ^(bulk)]) and shownin FIG. 1B. At pH 10.2, the mass transfer flux is approximately 35mmole/m²/h, which corresponds to biomass productivity of 20 g/m²/d(assuming a biomass C-content of ˜50%).

During cultivation, HCO₃ ⁻ is taken up, CO₂ is abstracted and fixed,resulting in a net release of OH⁻ as shown in Eqs. 7 and 8 below:

The production of OH⁻ shifts the DIC equilibrium towards CO₃ ²⁻ (see Eq.3) which, in turn, increases the driving force for CO₂ dissolution. Anynet increase in pH and associated decrease of HCO₃ ⁻ due to conversionto CO₃ ²⁻ can be rebalanced at night, when photosynthesis is absent (Eq.1).

Suitable alkaliphilic algae include, but are not limited to, eukaryoticmicroalgae such as Chlorella sp. and Dunaliella sp., as well ascyanobacteria such as Synechocystic sp., Cyanothece sp., Microcoleussp., Euhalothece sp., and Spirulina sp. Some non-limiting examples ofalkaliphilic algae strains include Synechocystis salina, Aphanothecestagnina, Chamaesi-phon subglobosus, Rhabdoderma lineare, Synechococcuselongates, Phormidium ambiguum, Phormidium foveo-larum, Phormidiumretzii, Oscillatoria splendid, Sscilla-toria limnetica, Spirulinafusiformis, and Spirulina laxissima. However, any algae that can thriveat high pH values (>9.5) and withstand high (˜60-100 mM) inorganiccarbon content can be utilized.

The multi-step process of DIC transport into alkaliphilic microalgaecells and ultimate conversion to organic carbon is shown in FIG. 2. Theconversion to organic carbon using light energy (i.e., photosynthesis)takes place via two parallel processes: light-dependent andlight-independent reactions (FIG. 2). The light-dependent reactionsoccur in the thylakoid region of cells with two photosystems (PS 1 andPS II) that work in tandem to produce energy (ATP) and reducingequivalents (NADPH). When a photon strikes chlorophyll within thephotosystem, the absorbed energy is either used to split H₂O (at PSII)and recover electrons (e⁻) or is dissipated as heat/fluorescence. Therecovered e⁻ are transported through an electron transport chain(through ferredoxins, Fd) and used for generation of reducingequivalents (NADPH) or for nitrate reduction and protein synthesis (seethe reactions below the dotted line, FIG. 2).

In parallel, under alkaline conditions, light-independent DIC uptakeoccurs via carbon concentrating mechanisms (CCMs) that consist of aseries of active HCO₃ ⁻ transporters, carbonic anhydrases, and, in somecases, conversions of C3 and C4 molecules (not depicted in FIG. 2 forsimplicity). Since multiple enzymatic steps are involved, the DICtransport process can be kinetically limited by external DICconcentrations. In the presence of high DIC, the net flux through thetransporters (shown as filled ovals in FIG. 2) is enhanced such thatmore DIC is available for carbonic anhydrases that can ultimatelydeliver CO₂ to RuBisCO at higher rates. The half-saturation constant forcarbonic anhydrases is generally in the 20 mM range, and low DICavailability severely limits the kinetics of this enzyme. In addition,the carbonic anhydrase gene expression is low under low HCO₃ ⁻concentrations, which may decrease the availability of CO₂ at RuBisCO.Such a decreased availability of CO₂ at RuBisCO would negatively impactthe rates of carbon fixation through the Calvin cycle. Simultaneously,the high cellular DIC flux drives the light-dependent reactions towardshigher production of NADPH by increasing NADPH demand for use in carbonfixation. Thus, by maintaining high alkalinity, higher photosyntheticefficiencies and ultimately high rates of CO₂ fixation can be achieved.

The mechanisms of inorganic carbon uptake from the atmosphere and usefor photosynthesis are (as described above) innately established innatural alkaline lakes which have the highest reported aquaticphotosynthetic carbon fixation rates. In addition to facilitatingsustained supply of CO₂ from the atmosphere (rather than flue gases),the use of high-pH and high-alkalinity media can enable the sustainedcultivation of desired species due to the relatively low microbialdiversity in these harsh environments. Grazer infestations are also lesslikely in alkaline environments. For example, Daphnia eggs loseviability when pH values exceed 10-10.5. In commercial practice,Spirulina production is successful, at least partly, due to the high pHgrowth conditions that enable prolonged maintenance of thesecyanobacterial species in low-cost open ponds. A SLA-04 culture crashhas not been observed despite several months of outdoor cultivation inhigh-pH and high-alkalinity media.

The adjustment of macro- and micro-nutrient concentrations results inimprovements in carbohydrate and lipid productivity. One of theprincipal macro-nutrients important for algae cultivation is nitrogen(N). N is also a significant contributor to the net carbon footprint ofalgal biofuels. Low N is also very desirable for downstream conversionprocesses since the resulting fuels also have a low N content.Therefore, the cultivation of alkaliphilic algae on low N media wasevaluated. The results showed that the high biomass productivities canbe maintained at lower N in the media, and the resulting biomass alsohas a low N-content. An increase in pigment production (e.g.,chlorophyll b) when cellular N content is high has been observed, whichcauses cultures to become “dark” and detrimental to light penetration.Overall, by adjusting media alkalinity and N supply, biomass with lowN-content can be produced.

The requirements for the micro-nutrients Ca and Mg have also beenevaluated, as the effects of these micro-nutrients are generallyunderappreciated in the art. Typically, these micro-nutrients are addedat a concentration level of 5-7 mg/L (Bold's medium). However, underalkaline conditions their solubility in the medium is diminished. Thesereduced dissolved nutrient concentrations can induce “nutrient-limitedstress” on the growing microalgae. It is known that N-starvationimproves lipid productivity in microalgae. Therefore, whethermicro-nutrient (Ca and Mg) limitations would also lead to improvedbiomass and lipid productivity during alkaliphilic microalgaecultivation was evaluated.

The impact of increasing medium salinity on biomass growth was alsoevaluated. Use of saline water (from oceans or from saline/brackishgroundwater sources) improves the sustainability of microalgaecultivation by decreasing the requirements of freshwater. The results,described in the examples herein, indicate that cultivation ofmicroalgae in high salinity media containing excess nitrogen and highbiomass concentrations (i.e., conditions that limit light penetrationinto cultures), increased the production of phycocyanin—a high-valuenutraceutical.

Recycled media can be used in the open ponds. In some embodiments, highconcentrations (for example, 100 mM) of bicarbonate/carbonate salts areadded to the culture media to provide high alkalinity Hence, the abilityto recycle and reuse the media is important to minimize the costsassociated with replenishing these salts, and other unused nutrients. Asdescribed in the Examples herein, post-harvest media can be re-usedwithout detrimental impact on biomass productivity.

The high pH media permits open-pond cultivation in “mixotrophic mode”without culture crash. In addition to facilitating sustained supply ofCO₂ from the atmosphere (rather than from flue gases), the use ofhigh-alkalinity and high-pH media can enable sustained cultivation ofdesired species, since it is likely that contaminating populations willbe less diverse at higher pH values. A culture crash of the alkaliphilicstrain SLA-04 has not been observed in the presently described method,despite a significant number of months of outdoor cultivation in high-pHand -alkalinity media. The extreme pH and alkalinity of the medium alsoallows for low-cost outdoor pond mixotrophic cultivation withsignificantly lower chance for bacterial contamination—mesophilic (<pH8.5) outdoor cultivation wood likely not be possible with mesophilicalgae.

It is understood that an open pond utilizing the methods describedherein can include any apparatuses or structures common in open pondalgae systems. For example, the open ponds may include paddle wheels orother water-moving devices usable to keep the algae circulating, as wellas electronic controls, pumps, pipes, sensors, and the like. Continuousmixing of algal cultures is preferred in order to prevent thermalstratification and cell sedimentation, and to maintain carbonation. Insome embodiments, the open ponds are known as raceway ponds, resemblinga race track. A typical open pond is about one-foot deep, from about oneacre to several acres in size, where the algae is exposed to naturalsolar radiation which is converted into biomass. An open pond system canbe constructed out of any suitable material for containing the medium,such as PVC, PE, or concrete. Further, one skilled in the art willrecognize that once the algae is harvested (such as by centrifugation),any method known in the art can be utilized to convert the harvestedbiomass to one or more high-value downstream products such as fuels,including hydrothermal liquefaction. In some embodiments, the biomassharvested from the open ponds as described herein can be converted tobiofuels with lower nitrogen content than algae from conventional openponds.

EXAMPLES

Example 1: Effect of HCO₃ ⁻ content on SLA-04

Biomass Growth and Productivity

The Chlorella sp. strain SLA-04 (henceforth referred to as SLA-04) wasisolated from Soap Lake in the State of Washington (USA). Cultures weregrown in a medium that comprises the nutrients: NaNO₃ (1.05 mM), KH₂PO₄(0.3 mM), MgSO₄.7H₂O (0.3 mM), CaCl₂.2H₂O (0.17 mM), NaCl (0.42 mM),ferric ammonium citrate (10 mg/L)), and 1 mL trace metal solution. Thetrace metal solution comprised H₃BO₃ (9.7 mM), MnCl₂.4H₂O (1.26 mM),ZnCl₂ (0.15 mM), CuCl₂.2H₂O (0.11 mM), Na₂MoO₄.2H₂O (0.07 mM),CoCl₂.6H₂O (0.06 mM), NiCl₂.6H₂O (0.04 mM), V₂O₅ (0.01 mM), and KBr(0.08 mM). For experiments that were started in a mildly alkaline pHmedium (8.7 and 8.2), NaHCO₃ ⁻ was added as an inorganic carbon sourceat HCO₃ ⁻ concentrations in the range of 7-40 mM. For pH-controlledcultures, pH was controlled by periodic CO₂ addition through asolenoid-regulated control system that maintained the pH at anapproximate value of 8.7 (Neptune Systems Apex, N.C., USA). (ExperimentA.) For experiments that were started under significantly higheralkaline pH conditions (pH 10), equal molar concentrations of NaHCO₃ ⁻and Na₂CO₃ were added to achieve final HCO₃ ⁻ concentrations of 4.5-30mM. (Experiment B.)

Open raceway ponds with dimensions of 2′×1′×1′ (L×W×D) were constructedand used in these experiments. These ponds were equipped with areal-time temperature and pH monitoring and data logging system (NeptuneSystems Apex, N.C., USA). The ponds were placed in a heated greenhouse.Tap water available at the greenhouse facility was first filteredthrough a 10 μm filter (to remove sediments) and then used for mediumpreparation. The working volume of the culture was kept at 5″ forExperiment A and 6″ for Experiment B.

During Experiment A, biomass concentrations (measured as cell dry weight(CDW) and productivity of cultures grown in media with varying HCO₃ ⁻concentrations and without pH control were assessed and compared withpH-controlled controls (FIGS. 3A-3B). The results show that biomassconcentrations as well as biomass productivity were greater when HCO₃ ⁻concentrations in the media were higher. Maximum biomass productivitywas observed to be 24.2±1.0 g CDW/m²/day for cultures that initiallycontained 40 mM HCO₃ ⁻. These productivity values were similar(23.4±0.28 g CDW/m²/day) to the productivities measured in pH-controlledcultures containing 20 mM HCO₃ ⁻ (FIG. 3A).

Because of CCMs, microalgae can accumulate HCO₃ ⁻ in cytosol andsubsequently deliver high CO₂ concentrations around theribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) enzyme, andthus increase the rates of photosynthetic carbon fixation (FIG. 2).However, the HCO₃ ⁻ transfer rate into cells is an important factor tomaintain high cytosolic HCO₃ ⁻ concentration as well as photosyntheticrate. Higher media concentrations of HCO₃ ⁻ lead to improved masstransfer rates and cellular uptake.

To assess the impact of HCO₃ ⁻ on photosynthetic efficiency (i.e., theefficient use of incident photons), “rapid light curve” measurementswere made. As shown in FIG. 2, only a portion of the electrons generatedfrom the incident light are utilized towards photosynthesis while therest are dissipated. The electrons participating in photosynthesis serveto electrochemically reduce cellular DIC to carbohydrates via NADPHmediated reactions at RuBisCO (FIG. 2). The flow of electrons towardsphotosynthesis is referred to as the “electron transfer rate” (ETR).Higher values of ETR were observed in cultures containing higherconcentrations of bicarbonate in the media (FIG. 3C). Cultures fed withhigh HCO₃ ⁻ content (40 mM) showed a maximum ETR value (ETR_(max)) of 32μmol/m²s, while cultures fed with 7 mM HCO₃ ⁻ showed much lowerETR_(max) values of 13 μmol/m²s (FIG. 3C). Overall, the higheralkalinity medium allowed better photon utilization.

pH change

An increase in pH was observed during algal growth at day time (lightcycle) due to uptake of bicarbonate and release of hydroxyl ions (Eq 7).pH decreased at night due to CO₂ release from microalgae respiration(FIG. 4A).

Example 2: Biomass Growth and Productivity in High-pH Media with VaryingLevels of Alkalinity

Biomass Growth, Productivity

Experiment B was performed to assess biomass productivity at high pH.Since Experiment A (FIGS. 3-4) showed improvement in productivity withhigher alkalinity, a similar approach was used in Experiment B and mediaalkalinity was varied. Experiment B was performed in media initially atpH 10. The alkalinity of the media was set such that initial bicarbonateconcentrations were 7, 20, and 30 mM. An experiment at an initial pH of8.2 and initial HCO₃ ⁻ concentration of 30 mM served as a control. FIGS.5A-5C depict the growth, productivity, and ETR of SLA-04 cultures duringExperiment B. As observed in Experiment A (Example 1), biomass growth,productivity, and ETR increased when HCO₃ ⁻ concentration in mediumincreased. The results revealed that with the same HCO₃ ⁻ availability(30 mM), cultures started with higher pH (9.9) showed higher growth aswell as maximum productivity (34.7±1.5 g CDW/m²/day) than culturesstarted with low pH (8.2) (FIGS. 5B, 5C). Table 1 (FIG. 13) shows thatwhen compared to low HCO₃ ⁻ conditions, the parameters related toefficient energy capture towards carbon fixation were higher, and theparameters related to energy dissipation were lower under high HCO₃ ⁻conditions.

TABLE 1 Rapid light curve parameters for cultures grown under high andlow HCO₃ ⁻ concentrations. High HCO₃ ⁻ Low HCO₃ ⁻ Description Parameter(30 mM) (7 mM) Energy capture Effective PS II quantum yield Y (II) 0.3610.209 towards carbon Maximum electron transfer rate ETRmax 17.9 9.4fixation (μmol/m²s) Photosynthetic efficiency (eL/ph.) α 0.167 0.098Light saturation (μmol/m²s) I_(k) 126 96 Energy Quantum yield ofregulated Y (NPQ) 0.066 0.094 dissipation energy dissipation Quantumyield of non regulated Y (NO) 0.573 0.697 energy dissipation

Nitrate utilization efficiency (g biomass/g nitrate utilized) wasobserved to be higher in cultures with high HCO₃ ⁻ availability (FIG.5D), resulting in low nitrogen content in the biomass. This is importantto produce low nitrogen content biofuels from biomass intermediatesthrough hydrothermal liquefaction (HTL). Moreover, it reduces the costand greenhouse gas emissions associated with industrial nitrateproduction.

pH Change, and Atmospheric CO₂ Capture

Change in pH during algal growth is illustrated in FIG. 6A. Cultures fedwith high HCO₃ ⁻ content showed higher buffering capacity than culturesfed with low HCO₃ ⁻ content. Detailed OH⁻ increase and controlledmechanisms are shown in Table 2.

TABLE 2 OH− ion balance of SLA-04 grown under different pH and inorganiccarbon conditions. Note: Increase in OH− concentration generated wascalculated from sum of increase in TOC and NO₃ ⁻ and phosphateutilization data. Increase in CO₃ ²⁻ indicates HCO₃ ⁻ to CO₃ ²⁻conversion. Atm. CO₂ absorption was calculated by: Atm. CO₂ absorption =OH⁻ generation - HCO₃ ⁻ to CO₃ ²⁻ conversion + ΔOH⁻ OH⁻ Controlmechanisms Inorganic Increase in Atm. CO₂ HCO₃ ⁻ to CO₃ ²⁻ Atm. CO₂absorption/ carbon OH⁻ conc. absorption conversion Δ OH⁻ HCO₃ ⁻ to CO₃²⁻ (mM) (mM) (mM) (mM) (mM) conversion  7 6.74 5.26 0.56 0.91 9.33 4012.39 6.06 6.10 0.23 1.00 60 13.44 7.62 5.70 0.12 1.34 30 pH 8.2 11.352.46 8.77 0.12 0.28

The data indicate that with the same initial inorganic carbonavailability (60 mM), cultures started with pH 9.9 showed higher (1.34)atm. CO₂ absorption/HCO₃ ⁻ to CO₃ ²⁻ conversion than cultures startedwith pH 8.2 (0.28). These results indicate that under high pH algalgrowth conditions, atmospheric CO₂ absorption dominates over HCO₃ ⁻ toCO₃ ⁻ conversion and results in low inorganic carbon drain. Carboncontent of dried biomass was observed to be in the range of 44-47%. FIG.5B depicts the carbon balance data for all cultures. Maximum ΔTOC(12.3±0.7 g C/m²) was observed in cultures fed with high inorganiccarbon content (60 mM). For these cultures, the inorganic carbon drainΔIC was observed to be 4.75±0.13 g C/m², revealing that the remaining61% (7.55±0.61 g C/m²) of biomass carbon was derived from atmosphericCO₂ absorption (FIG. 6B). The Example 1 and 2 results demonstrate thatincreasing initial pH from 8.7 to 9.9 results in an increase inatmospheric CO₂ fixation in biomass from 37% to 61%.

Example 3: Phototrophic and Mixotrophic Cultivation of Microalgae UnderHigh pH and Alkalinity

Sustainability of microalgal cultivation under phototrophic andmixotrophic conditions was studied in 1100 L ponds with efficient mixingby paddle wheel (Commercial algae Professionals, NC, USA) with a workingvolume of 750 L and a depth of 7″ in outdoor raceway ponds under high pH(˜10) and high inorganic carbon (˜100 mM) conditions without CO₂supplementation. Under the phototrophic conditions, cell dry weight, andbiomass and lipid productivities, were determined to be 23 g/m²/day and2 g/m²/day, respectively (FIG. 8A). The results indicate that ˜95% ofthe biomass carbon was derived from atmospheric CO₂, rather than fromthe added inorganic carbon (FIG. 8B). This shows that when compared toExample 2, the results demonstrate that efficient mixing of the culturemedium improved atmospheric CO₂ derived biomass carbon from 61% to 95%.Under mixotrophic cultivation with glucose, SLA-04 cultures grew withoutany measurable signs of contamination and showed significantly higherbiomass productivity (57 g/m²/day) due to the availability of additionalorganic carbon and associated reducing equivalents (FIG. 9). Lipidproductivities were also higher under mixotrophic conditions (7g/m²/day) relative to phototrophic SLA-04 cultures (2 g/m²/day) (FIG.9).

Example 4: Remnant Media Nutrients Recycling

After growth, remnant media was recovered by harvesting the algalbiomass through centrifugation. Then, the effect of remnant media (whichcontain high amount of inorganic carbon (˜60 mM)) on biomass growth wasevaluated by adding the used portion of nutrients only. The remnantmedia was recycled 8 times without any deleterious effects on algalbiomass growth. FIG. 10 depicts a comparison between algal biomassgrowth pattern using remnant media and fresh media.

Example 5: Nitrogen Utilization by SLA-04 and the Effect of N Input onSLA-04 Biochemical Composition

Nitrogen is a macronutrient and N content in biomass can determine theend-use of microalgae. For instance, high N-content (i.e., high protein)is desirable for microalgae use as food/feed ingredient. However, forbiofuel production low N in biomass is desirable since presence of N infuel is detrimental to fuel quality. Conventional cultivation methodsuse high concentration of N in the medium, which leads to production ofbiomass with high N content. The concentration of these nitrogenouscompounds in the biomass can be decreased by growing microalgae undernitrogen limitation conditions. But severe nitrogen limitation can alsoimpair growth. It was demonstrated that by maintaining an optimalconcentration of N in the media, the N-content of biomass can bedecreased without significant detrimental impact on biomassproductivity.

The results from 450 mL e-PBR experiments (FIG. 11) show that the highbiomass productivities can be maintained at lower N in the media. Theresulting biomass also has a low N-content (FIG. 11).

Additional indoor experiments were performed with SLA-04 cultures grownin 3 L reactors. Cultures were grown in a medium that comprised thenitrogen concentration in the range of 5-15 mg/L using NaNO₃ as anitrogen source. NaHCO₃ and Na₂CO₃ were added in a molar ratio of 2:3 toget a final HCO₃ ⁻ concentration (30 mM) and initial pH 10.1. Culturesadapted to high media N input (27 mg/L) with initial nitrogen content inbiomass about 7% was used as an inoculum. The reactors were placed on astir plate and illuminated by a bank of 4 Ecolux Starcoat 54 Wfluorescent tubes (GE Lighting, Cleveland, Ohio) on each side. Lightcycle was maintained at a PAR intensity ˜400 μmol/m²/s on each side for10 h.

The results (3 L reactors) show that the high biomass productivities canbe maintained even at N content 5 mg/L in the media (FIG. 13A) becausethe low N availability avoids excess production of nitrogen storagecompounds (e.g., for pigments). Also, an increase in chlorophyll pigmentproduction (FIG. 13B) was observed when medium nitrate content was high,which causes cultures to become “dark” and detrimental to lightpenetration. The results indicate all the cultures werephotosynthetically active, however there is no significant difference inphotosynthesis efficiency (F_(v)/F_(m)) and maximum electron transferrate (ETR_(max)) when N availability for the culture is decreased (Table3).

TABLE 3 Rapid light curve parameters for cultures grown in 3 L reactorsand under different NO₃ concentrations (time = 2 days). Media NETR_(max) PSII input (mg/L) Fv/Fm (μmol/m²s) 5 0.649 17.6 10 0.685 17.715 0.711 19.1 Note: Fv/Fm: maximal PS II quantum yield; ETR_(max) PSII:Maximum electron transfer rate (μmol/m²s).

Example 6: Nitrogen Utilization by SLA-04 and the Effect of N Input onSLA-04 Biochemical Composition—Outdoor Experiments at 30 L Scale

The outdoor experiment was conducted as a follow-up experiment to theindoor experiment with the same media conditions to examine theapplication of low-N, high-productivity cultivation in open ponds.Initial media N input was adjusted to a range of 5-27 mg/L using sodiumnitrate as a nitrogen source. In contrast to the indoor experiments,cultures were first adapted to experimental nitrogen conditions for tenbatches to get constant N content in biomass relative to the media Ninput. Then the experiments were conducted in open raceway ponds (30 L)with working volume of 20 L and performed in sequential batches, witheach batch lasting for a duration of two days.

FIG. 14A depicts biomass productivity of SLA-04 on media N inputconditions. The results show when the media N input increased, there wasan increase in biomass productivity (FIG. 14A). Since chlorophyll is thenitrogenous compound among others, as media N concentrations increases,there is an increase in chlorophyll content (FIG. 14C). Hence, whenconsidering chlorophyll concentration (FIG. 14C), the biomassproductivity of SLA-04 per chlorophyll content was observed to decreasealong with an increase in media N input (FIG. 14B). Without wishing tobe bound by theory, it is believed this is due to the light limitationto chlorophyll present in cells being present at greater pond depthsthat results in a decrease in biomass productivity relative tochlorophyll content. As the N input increases, there is an abundantavailability of nitrogen source and cultures tend to accumulationnitrogenous compounds (e.g., chlorophyll and related proteins). Thisresults in increased nitrogen content in biomass (range: 3.5-7.2%) (FIG.14D). F_(v)/F_(m) factor, which represents PSII maximum quantum yield atdark adapted state for cultures, is shown in FIG. 14B. Among othernutrients, nitrogen stress for culture (that damages PSII) decreasesF_(v)/F_(m). The results show F_(v)/F_(m) is increased in increase inorder with media N content (5-27 mg/L). Even though F_(v)/F_(m) ofcultures fed with low N (5-8 mg/L) had low F_(v)/F_(m), it did not showa significant effect on biomass growth and productivity (FIG. 14A).Nitrogen stress condition in microalgae can inhibit further cellreproduction and induce lipid synthesis as an energy storage process.Fatty acid methyl esters (FAME) data indicate that the cultures fed at Ninput 5 mg/L had four folds higher FAME content (16%) than cultures fedat N input 27 mg/L (4.1%) (FIG. 14F). Low F_(v)/F_(m) is the indicatorof stress condition for cultures fed with low (5 mg/L) N input prone toinduce lipid biosynthesis and thereby shown increased FAME content.Table 4 depicts the biochemical composition of SLA-04 fed at different Ninput. The results confirm one can modulate the biochemical compositionof the microalgae by regulating media N input.

TABLE 4 Biochemical composition of SLA-04 grown under different N inputenvironment Carbohydrate Moisture Sample Name (%) Ash (%) Protein (%)Fame (%) (%) Total  5 mg/L N-Batch 1 33.15 6.47 22.724 17.34 6.8 86.48 8 mg/L N-Batch 1 30.49 6.70 35.88 6.20 7.58 86.85 11 mg/L N-Batch 115.00 6.30 42.458 7.00 7.93 78.69 27 mg/L N-Batch 1 13.88 8.50 43.0564.00 8.03 77.47  5 mg/L N-Batch 3 36.31 5.75 22.724 16.56 6.39 87.73  8mg/L N-Batch 3 27.91 5.20 33.488 6.00 7.73 80.33 11 mg/L N-Batch 3 13.406.50 43.056 4.00 8.2 75.15

Example 7: Nitrogen Utilization by SLA-04 and Effect of N Input onSLA-04 Biochemical Composition—Outdoor Experiments at 1100 L Scale

Based on the above experiment, it is important to start cultures withthe same chlorophyll concentration to evaluate the effect of media Ninput on biomass production. The initial chlorophyll concentration wasadjusted to a similar concentration by appropriate dilution of inoculumfor all N input culture conditions. The experiments were conducted inbig raceway ponds (1100 L) with working volume of 500 L and at ˜5inches' depth. Initial media N input was adjusted to a range of 5-15mg/L using sodium nitrate as a nitrogen source.

Cultures fed with N concentration 5 mg/L showed higher biomassproductivity than cultures fed with N concentration 10 and 15 mg/L(FIGS. 15A-15B). The results indicate the biomass productivity increasesas the N input decreases in media because of two factors: i) high cellconcentration at N limitation conditions provide more cellular machineryfor carbon fixation, and ii) as the growth proceeds, the chlorophyllcontent of cultures supplemented with high N availability increases,prone to light limitation to deeper layers when compared with culturessupplemented with low N input.

The presence of high media alkalinity also decreases the N uptake bySLA-04 (FIG. 5D). Overall, these results show that by adjusting mediaalkalinity and N supply, biomass with low N-content (˜3%) can beproduced. Cultures grown outdoors (FIGS. 8A, 9) also had an N-content of˜3%. Most other microalgae have an N content of 4-8%.

Example 8: Optimization of Micro-Nutrient Utilization by SLA-04

Effect of Ca and Mg on Growth of SLA-04

It was observed that biomass and lipid productivities can be improved(up to 33%) through use of low concentrations of Ca and Mg (<1.5 mg-Ca/Land <0.5mg-Mg/L) (FIG. 12). Most microalgae growth media recipes havemuch higher Ca and Mg. For example, Ca and Mg concentrations in Bold'smedium are nearly 7 mg/L. This finding—that under alkaline conditions,it is possible to achieve higher biomass productivities and lipidaccumulation at significantly lower levels of Ca and Mg compared totraditional Bold's medium—has very favorable economic implications inlarge-scale outdoor algae cultivations.

Effect of Salinity (NaCl) on Growth of SLA-04

Lowering fresh water requirements is important for sustainablemicroalgae cultivation. Saltwater is a more sustainable water sourcethan fresh water. For instance, seawater is inexpensively accessible incoastal areas of the southeast US (e.g. Florida and other Gulf states)and brackish water is abundant in southwest US (e.g. Arizona, NewMexico, Texas). These locations also have the most appropriate weatherfor microalgae cultivation. Str. SLA-04 can thrive in high salinitymedia as it was isolated from saline-alkaline lake (Soap Lake, State ofWashington).

Growth of the isolated strain C. sorokiniana str. SLA-04 was examined ina BG-11 medium with nitrate content 40 mg/L and similar salinity toseawater (30 g/L). An appropriate proportion (2:3) of NaHCO₃ ⁻ andNa₂CO₃ ²⁻ were added as an inorganic carbon source to get a final HCO₃ ⁻concentration (30 mM) and initial pH 10.1. The experiment was performedin 3 L reactors under 800 μmoles/m²/s light illumination and light-darkcycles of 10 h/14 h.

Interestingly, the results show improvement of biomass productivity ofSLA-04 with medium containing high salinity (FIG. 16A). Such improvementcan be assigned to three factors: 1) increased availability of sodiumions, which are required for pH homeostasis of alkaliphilic C.sorokiniana str. SLA-04; 2) the Henry constant for dissolution of CO₂ inwater increases by increase in salinity, which improves carbon capturingand inorganic carbon availability for salty cultures; and 3) there is alower pigmentation of microalgae cells grown in saltwater than freshwater, which therefore increase light availability. F_(v)/F_(m) factor,which represents PSII maximum quantum yield at dark adapted state forindoor cultures, is shown in FIG. 16B. Any environmental or nutritionalstress for culture (that damages PSII) decreases F_(v)/F_(m). Theresults show there is no significant difference in F_(v)/F_(m) betweencultures grown with salinity and cultures grown without salinity.

The outdoor experiment was conducted with the same media conditions asthe indoor experiment to examine the application of this method for morecost-efficient and manageable open ponds. To evaluate the effect of saltconcentration, two different salt concentrations (18 g/L and 30 g/L)were used. The experiments were conducted in open raceway ponds (30 L)with working volume of 18 L, and were performed in sequential batchesthat each lasted 2 days.

FIG. 17A and FIG. 17B illustrate the growth pattern and biomassproductivity of SLA-04. The results indicate biomass productivity isincreased on the order of salt concentration increases in the medium(FIG. 17A). Chlorophyll a is the pigment that interacts directly in thelight requiring reactions of photosynthesis, whereas chlorophyll b is anaccessory pigment and acts indirectly in photosynthesis by transferringlight it absorbs to chlorophyll a. Excessive accumulation of chlorophyllb limits the light penetration in to deeper layers. Low lightavailability further decrease photosynthetic activity and increaserespiration rates, and is thereby attributed to low productivity. Thelower chlorophyll concentration and higher chlorophyll a/b ratio of thecultures is the indication of efficient light penetration andphotosynthetic efficiency for the cultures with high salinity (FIGS.17C-17D). The Henry constant for dissolution of CO₂ in water increasesby the increase in salinity, which results in higher atmospheric carboncapture under salinity conditions (Table 5). Overall, these results arein accordance with indoor experiments and indicate that saltwater can beused as an inexpensive water source to enhance the biomass productivityand carbon capturing.

TABLE 5 Carbon balance of SLA-04 grown under different salinityconditions Assimilated organic Inorganic carbon Atmospheric carboncarbon (mM) depletion (mM) captured (mM) 0 g/L 18 g/L 30 g/L 0 g/L 18g/L 30 g/L 0 g/L 18 g/L 30 g/L Batch 1 5.3 6.7 7.4 0.3 0.5 0.4 5 6.1 7Batch 2 4.5 4.3 6.1 0.6 0.1 0.3 3.9 4.2 5.8

Effect of Salinity on Phycocyanin Production

Phycocyanin is a light-harvesting pigment and nitrogen-storing proteinfound in the prokaryotic cyanobacteria species, as well as in eukaryoticmicroalgae. Phycocyanin is widely used in pharmaceuticals and bluepigments. It is used as a natural dye for foods and cosmetics. Chlorellasorokiniana is one of the highest natural sources of phycocyanin andchlorophyll. Hence, the strain C. sorokiniana str. SLA-04 has theability to produce phycocyanin. Environmental stresses such as lightintensity, culture concentration, salinity, pH, and nitrogenavailability can influence phycocyanin production in microalgae. In thisexample, the effect of salt concentration, inoculum concentration, andnitrogen content on phycocyanin production of C.sorokiniana str. SLA-04was evaluated.

Culture conditions: since phycocyanin is the nitrogen storage compound,when compared to the above-described outdoor experiment, the mediumnitrate concentration was increased from 40 mg/L to 150 mg/L to providenitrogen abundant environment. Also, inoculum concentration wasincreased from 0.32 to 0.75 g/L.

FIGS. 18A-8B depict growth and average biomass productivity of theSLA-04 under the culture conditions. In contrast to the above-describedexperiments, the results show there is no significant difference inbiomass growth and productivity between cultures grown with and withoutsalinity. Without wishing to be bound by theory, it is believed this isdue to the high pigmentation noticed in saline cultures (FIGS. 18C-18D).A comparative analysis between batch 1 and batch 2 cultures indicatedthat cultures started with low inoculum concentration showed higherbiomass productivity. This is attributed to better light penetration.

FIGS. 18C-18D show pigmentation (phycocyanin and chlorophyll) of SLA-04under culture conditions. The results show salt stress increased thephycocyanin content in biomass. As the salt concentration increases,phycocyanin content in biomass was observed to be increased in order(FIG. 18C). Like the above-described experiments, the results revealed adecrease in total chlorophyll content and an increase in chlorophyll a/bratio with respect to salt concentration in the medium (FIGS. 18D-18E).

Certain embodiments of the methods and systems disclosed herein aredefined in the above examples. It should be understood that theseexamples, while indicating particular embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseexamples, one skilled in the art can ascertain the essentialcharacteristics of this disclosure, and without departing from thespirit and scope thereof, can make various changes and modifications toadapt the compositions and methods described herein to various usagesand conditions. Various changes may be made and equivalents may besubstituted for elements thereof without departing from the essentialscope of the disclosure. In addition, many modifications may be made toadapt a particular situation or material to the teachings of thedisclosure without departing from the essential scope thereof.

What is claimed is:
 1. A method for culturing algae, the methodcomprising: culturing alkaliphilic algae in an open pond medium having apH above 9.5, sufficient to allow increased fixation of atmospheric CO₂into the open pond medium; incorporating into the open pond medium aninorganic carbon buffer; and incorporating into the open pond medium Caat a concentration of less than 1.5 mg/L and Mg at a concentration ofless than 0.5 mg/L; wherein the open pond medium is free of anyconcentrated supply of CO₂, and no concentrated source of CO₂ is used tosupply carbon into the open pond medium.
 2. The method of claim 1,wherein the inorganic carbon buffer comprises either a NaHCO₃/Na₂CO₃mixture or a KHCO₃/K₂CO₃ mixture.
 3. The method of claim 2, wherein theNaHCO₃/Na₂CO₃ mixture or the KHCO₃/K₂CO₃ mixture is incorporated intothe open pond medium at a concentration ranging from about 7 mM to about1 M.
 4. The method of claim 1, wherein the pH of the open pond medium isat least about 9.9.
 5. The method of claim 1, further comprisingincorporating glucose or other sugars or carboxylic acids into the openpond medium.
 6. The method of claim 1, wherein the algae compriseseukaryotic microalgae or prokaryotic cyanobacteria.
 7. The method ofclaim 1, further comprising circulating the algae within the open pondmedium.
 8. The method of claim 1, further comprising harvesting biomassfrom the cultured algae and recovering remnant media.
 9. The method ofclaim 8, further comprising recycling the remnant media in a second openpond medium.
 10. The method of claim 8, further comprising convertingthe harvested biomass to one or more fuels.
 11. The method of claim 10,wherein the converting comprises hydrothermal liquefaction to producebiocrude having a N content of less than 4%.
 12. The method of claim 1,further comprising regulating nitrogen input in the open pond medium, ina range from about 5 mg/L to about 27 mg/L, so as to modulate thebiochemical composition of the microalgae.
 13. The method of claim 1,further comprising improving phycocyanin production by increasing one ormore of biomass concentration, nitrogen concentration, and salinity inthe open pond medium.
 14. A method for culturing algae, the methodcomprising: culturing alkaliphilic algae in an open pond medium having apH above 9.5, sufficient to allow increased fixation of atmospheric CO₂into the open pond medium; incorporating into the open pond medium aninorganic carbon buffer; and regulating nitrogen input in the open pondmedium, in a range from about 5 mg/L to about 8 mg/L, so as to modulatethe biochemical composition of the algae; wherein the open pond mediumis free of any concentrated supply of CO₂, and no concentrated source ofCO₂ is used to supply carbon into the open pond medium.